Polygala fallax, a member of the family Polygalaceae, grows as a shrub or small tree with stout roots, gray-green branchlets, lanceolate to elliptic-lanceolate leaves and racemes of yellow flowers. It is endemic to southern China, occurring in Fujian, Guangdong, Guangxi, Guizhou, Hunan, Jiangxi and Yunnan Provinces, typically in forests, along streamsides and in moist valley habitats at altitudes of 400 to 1700 meters. In traditional Chinese medicine it has long been used to treat lumbar and leg pain caused by strain, as well as acute and chronic hepatitis. Its main active components, including flavonoids, polysaccharides, volatile oils and saponins, underpin a growing body of pharmacological research pointing to effects ranging from lipid lowering and anti-hepatocellular carcinoma activity to improved recovery of liver tissue and relief of myocardial ischemia. Recent work has even suggested the plant may delay aging in rats by down-regulating the phosphorylation of PI3K, AKT, mTOR and S6K1, key proteins in the PI3K/AKT/mTOR signaling pathway.

Despite this medicinal promise, cytological and genomic research on P. fallax has remained strikingly limited. Most existing studies focus on its pharmacological components and activities, leaving a substantial gap in basic genetic knowledge. To fill that gap, a research team based at the South China Botanical Garden of the Chinese Academy of Sciences combined three complementary approaches: k-mer analysis of high-throughput sequencing data, flow cytometric measurement of nuclear DNA content, and classical chromosome counting from root tip meristems. Together, these methods provide a reliable first portrait of the species’ genome before any attempt at full assembly.

For the genome survey, the researchers collected fresh leaves from plants originally gathered in natural populations in Ruyuan County, Guangdong Province, at an altitude of 890 meters, and cultivated at the botanical garden. Genomic DNA was extracted using the CTAB protocol and sequenced on an Illumina NovaSeq 6000 platform, generating approximately 50-fold coverage of the estimated genome. After trimming adapters and filtering for quality, 109.79 gigabases of clean data remained from 110.40 gigabases of raw sequence, with more than 99 percent of bases at Q20 quality and over 97 percent at Q30. The genome’s GC content was measured at 38.3 percent, a moderate value unlikely to introduce GC bias during sequencing and assembly.

The heart of the genome survey lay in k-mer analysis. Using Jellyfish to count 19-mers and GenomeScope 2.0 to model their frequency distribution, the team tallied more than 85.5 billion k-mers. The resulting distribution curve showed a main peak at a depth of 39.2, a heterozygosity peak near 19 to 20, roughly half the main peak’s coverage, and a duplication peak near 80, roughly double the main peak, a pattern consistent with a diploid genome. The tailing of the duplication peak signaled the presence of abundant repetitive sequences and a relatively large genome. From these data, the researchers estimated a genome size of approximately 2199.64 megabases, or about 2.20 gigabases, a heterozygosity rate of 1.11 percent, and a repeat content of 73.61 percent, notably high for a plant genome.

Flow cytometry provided an independent check on genome size. Fresh leaf tissue from P. fallax was chopped together with leaf material of tomato, Solanum lycopersicum, whose 2C-DNA value of 0.90 gigabases served as an internal reference standard. Nuclei were stained with propidium iodide and measured on a BD FACSCalibur flow cytometer with 488 nm blue light excitation, with 10,000 particles counted per run and coefficients of variation kept within 5 percent. The fluorescence intensity ratio of P. fallax to tomato was 2.77, yielding a 2C-DNA value of approximately 2.49 gigabases. This figure is slightly larger than, but close to, the k-mer-based estimate, a convergence the authors say supports the reliability of both measurements.

Chromosome counting completed the cytogenetic picture. Young root tips, one to two centimeters long, were collected from five individual plants between 10 and 11 in the morning, pretreated with 0.01 percent aqueous colchicine for six hours, fixed in a 3:1 mixture of absolute ethanol and glacial acetic acid, hydrolyzed in 1M hydrochloric acid at 60 degrees Celsius for 50 minutes, and stained with carbol fuchsin before squashing. Examination of 20 metaphase plates under an Olympus BX-43 microscope at 100 times magnification consistently revealed 40 chromosomes in every cell. The somatic chromosome number of P. fallax is therefore 2n = 2x = 40, with a primary basic number of x = 20, reported here for the first time.

That chromosome count turns out to be genuinely unusual. Within the genus Polygala, the largest in the family Polygalaceae with roughly 600 taxa worldwide, chromosome numbers are remarkably diverse, with reported somatic counts including 2n = 14, 16, 24, 28, 30, 32, 34, 36, 38, 40, 42, 46, 52, 56, 68, 72 and 96. Four primary basic numbers, x = 6, 7, 8 and 10, plus secondary bases of 17, 19 and 23, have been documented. A check of the Chromosome Counts Database revealed that only one other species, P. curtissii, shares the 2n = 40 count of P. fallax, underscoring how distinctive this endemic Chinese species is among its relatives.

The genome size story is equally intriguing. Among the 13 Polygala species with recorded genome sizes, P. fallax ranks third largest, at roughly three times the size of P. tenuifolia, the only member of the family with a published genome, assembled at 769.62 megabases. Yet P. fallax has only two more chromosomes than P. tenuifolia, which carries 2n = 38. The comparison highlights a well-known principle in plant genomics: chromosome number and genome size are not tightly correlated. P. paucifolia, for instance, holds the largest genome in the genus at 3.03 gigabases while sharing the same chromosome number, 2n = 34, as P. amara subsp. brachyptera, whose genome is among the smallest. Similar patterns appear elsewhere, as in the Caryophyllaceae, where Silene latifolia and S. vulgaris are both diploid with 2n = 24 yet differ markedly in genome size and mode of reproduction.

What drives such large genomes? The authors point to whole-genome duplication events and the accumulation of repetitive sequences, particularly tandem repeats and transposable elements, which can replicate and insert themselves throughout the genome, inflating its size without changing chromosome counts. Ancient polyploidy followed by diploidization is another well-supported route to large genomes in flowering plants, though this phenomenon has not yet been reported in Polygalaceae. Until the publication of the P. tenuifolia genome in 2023, no chromosome-level reference genome existed for any member of the family, a data gap that has hampered comparative genomics, gene family analysis and evolutionary studies. The new baseline data for P. fallax should help change that. Because whole-genome duplications and tandem duplications have been shown to drive the expansion of P450 and UGT gene families central to the synthesis of triterpenoid saponins, the main medicinal components of P. fallax, understanding how its large genome formed could illuminate the mechanisms behind its pharmacologically active compounds. The study’s genomic and cytological data now provide an important reference for selecting assembly strategies in future whole-genome sequencing, and for research on species conservation and the sustainable use of this over-collected medicinal herb.

Subject of Research: Genome size estimation and chromosome number determination of the endemic Chinese medicinal plant Polygala fallax

Article Title: Genome survey and chromosome number determination of Polygala fallax ( Polygalaceae ), an endemic medicinal plant from southern China

Article References: Fu, L., Liu, Z., Han, L., Zhang, T., Jiang, Y., Krivenko, D. A., Hu, J., Chen, H., & Duan, L. (2026). Genome survey and chromosome number determination of Polygala fallax (Polygalaceae), an endemic medicinal plant from southern China. Comparative Cytogenetics, 20, 127-139. https://doi.org/10.3897/compcytogen.20.184176

Image Credits: AI Generated

DOI: 10.3897/compcytogen.20.184176

Keywords: Polygala fallax, Polygalaceae, genome survey, k-mer analysis, flow cytometry, chromosome number, genome size, medicinal plant, heterozygosity, repetitive sequences, traditional Chinese medicine, cytogenetics